DNA strand breaks occur as intermediates in pathways of DNA replication, DNA repair and genetic recombination. The ability to join interruptions in the phosphodiester backbone is critical for genome integrity and stability. Three human genes encoding DNA ligases have been identified. There is compelling genetic and biochemical evidence linking the product of the human LIG1 gene, DNA ligase I, with DNA replication and DNA excision repair. Moreover, the reiteration of a mutation identified in the LIG1 gene of a human individual, whose symptoms included developmental abnormalities, immunodeficiency and lymphoma, in a mouse model has proven that DNA ligase I deficiency causes genetic instability and predisposition to cancer. Previously we have characterized a physical and functional interaction between human DNA ligase I and the replicative sliding clamp, PCNA. Since this interaction did not stimulate DNA joining, we searched for additional factors that may act at the ligation step. In preliminary studies we have detected a physical and functional and functional interaction with the replicative clamp loader RFC and shown that similar interactions occur among the functionally homologous yeast proteins, Cdc9 DNA ligase, yRFC and yPCNA (Pol30). In Specific Aim 1, we will elucidate the moelcular mechanisms by which these pairwise interactions co-ordinate Okazaki fragment joining. In Specific Aim 2, we will examine of phosphorylation in regulating the functional interaction between DNA ligase I and RFC. We have also found that DNA ligase I interacts with an alternative clamp loader complex containing hRad17 and an alternative clamp, hRad9-hRad1-hHus1 complex, suggesting a novel and unexpected link between DNA ligase I and cell cycle checkpoints activated by DNA damage and replication block. In Specific Aim 3, we will characterize the interactions between DNA ligase I and the checkpoint clamp loader/clamp complexes. The long term goal of these studies is to understand the molecular mechanisms by which DNA ligase I functions in DNA replication and repair.